Mature T cell blasts are exquisitely sensitive to antigen-MHC complexes, able to detect even a single molecule of ligand on the surface of an antigen presenting cell. We propose to extend our survey of mature T cell sensitivities and thresholds for activation to include naive cells, memory cells, regulatory T cells and Th subsets. We are also developing an assay to measure the peptide-MHC ligand requirements for cytokine secretion in these cells as well. We will also compare the peptide sensitivities of immature thymocytes for cell death with those of mature cells for activation. These differences in sensitivity or threshold should shed light on how T cell selection in the thymus and in the periphery maximizes sensitivity to foreign antigens but minimizes self reactivity. We will also continue our work on the mechanism of this remarkable T cell sensitivity to antigen, where our preliminary data indicates that endogenous peptide-MHC's play an important role in synergizing with agonist ligands. We will pursue studies to confirm and extend our results with soluble heterodimers to cell surfaces and to an artificial membrane system. We will employ fluorescence energy transfer between agonist and endogenous ligands on artificial membranes, both in bulk and in single molecule studies. We will also compare the structures of these endogenous pMHC's to agonists to discern the 'rules' that may govern their activity (or lack thereof). We will also investigate the role of the endogenous peptides that we find out is 'co-agonists' in the periphery to see if they have a role in thymic positive selection. Finally, we are collaborating with the Hesselink lab to develop new and more sensitive methods of surface plasmon resonance in order to measure the binding of endogenous peptide-MHC complexes to TCR's, something which current technology is unable to do. This promises to give important clues as to why some of these ligands act as co-agonists whereas others do not.